3-Nitrobenzanthrone (3-NBA) has been isolated from diesel exhaust and airborne particles and identified as a potent direct-acting mutagen in vitro and genotoxic agent in vivo. In order to evaluate the in vivo toxicity and carcinogenicity of 3-NBA in a situation corresponding to inhalation, a combined short-term and lifetime study with intratracheal (i.t.) instillation in female F344 rats was performed. DNA adduct formation, as a marker for the primary effect and analyzed by 32P-HPLC after single instillation, showed a few major DNA adducts and a rapid increase with a peak after 2 days, followed by a decline. No DNA adducts above the background level were observed after 16 days. The highest DNA adduct formation was observed in lung [approximately 250 DNA adducts/10(8) normal nucleotides (NN)] closely followed by kidney (approximately 200 DNA adducts/10(8) NN), whereas liver contained only 12% (approximately 30 DNA adducts/10(8) NN) of the levels of DNA adducts found in lung. In the tumor study, squamous cell carcinomas were found after 7-9 months in the high-dose group (total dose of 2.5 mg 3-NBA) and after 10-12 months in the low-dose group (total dose of 1.5 mg 3-NBA). The fraction of squamous cell carcinoma out of the total amount of tumors observed at the end of experiment at 18 months, corresponded to 3/16 and 11/16 in the low- and high-dose group, respectively. A single case of adenocarcinoma was also observed in each group. In the control group, no tumors were observed during the entire study of 18 months. In addition, a few cases of squamous metaplasia were also observed in the lung in both dose groups but not in the controls. In conclusion, 3-NBA forms DNA adducts in the lung immediately after i.t. administration but almost all DNA adducts were eliminated after 16 days. Tumor formation in two dose groups was observed in a dose-dependent manner with squamous cell carcinomas as the predominant tumor type at high exposure.
A further development of an HPLC method to analyze 32P-postlabeled DNA adducts is presented. The method is based on on-line detection of 32P radioactivity after separation by reversed-phase chromatography. The method has an advantage in that the postlabeling mixture can be injected directly into the HPLC system without any prior purification, with the background radioactivity on a low level. The analysis includes the whole range of substances from orthophosphate to non-polar DNA adducts, which makes it possible to analyze normal nucleotides and ATP together with DNA adducts. The analytical system has a high reproducibility and separates complex mixtures of DNA adducts. The slightly lower sensitivity compared to the TLC method is compensated for by the possibility of injecting large amounts of DNA into the system without affecting the analytical properties. The system can be applied to different DNA adducts as well as complex mixtures of DNA adducts.
Modifications at two points in the sequence of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis have contributed to a more accurate and simplified determination of 8-OH-dG in DNA. The first was an improvement in the detection limit for 8-OH-dG in high-performance liquid chromatography analysis and the second was a pronase digestion and ethanol precipitation method (pronase/ethanol method) for DNA isolation which could minimize artificial formation of 8-OH-dG. Since the changes in background current from electrochemical detection are regularly periodical, it was possible to reduce this background change by connecting a pressure damper, degassing the eluent before use and finally subtracting its theoretical function. After this background correction, the detection limit for 8-OH-dG was improved one order of magnitude, from 20 fmol (5.68 pg) to 1.76 fmol (0.5 pg). Therefore, 0.005 8-OH-dG/10(5) dG can be detected from 50 micrograms DNA. This improvement will allow the analysis of small samples, tissues from needle biopsies, < 5 ml whole blood, etc., and will contribute to the accuracy of 8-OH-dG measurements. The pronase/ethanol method resulted in lower levels of 8-OH-dG than the phenol method in analyses of both rat liver and calf thymus DNA, even after 6 h incubation at 45 degrees C. The level obtained by the pronase/ethanol method with butylated hydroxytoluene was approximately equal to or lower than the 8-OH-dG levels reported in normal rat liver. The pronase/ethanol method for DNA isolation can replace the phenol or other methods in 8-OH-dG analysis. This method also omits the use of highly toxic organic solvents.
DNA adducts have been detected in laboratory animals after exposure to carcinogens as well as in human populations with known or suspected risk of developing cancer. Examples are smokers, coke and aluminium workers, urban citizens and roofers. The formation of DNA adducts is an early event in carcinogenesis which can be used for measuring target dose and as a biomarker for genotoxic risk. A method of analyzing 32P-postlabelled DNA adducts on reverse HPLC with on-line detection of 32P has been developed. The method permits direct injection of the 32P-postlabeling mixture into the analytical system without prior purification with background radioactivity on a low level. The method can be used in parallel with TLC analyses of 32P-postlabelled DNA adducts to improve the analytical capacity. The time for analysis of a typical single sample by HPLC and TLC is 30-60 min and 6-24 h respectively. A high (2 M) salt concentration in the HPLC eluent reduces the 32P background considerably. Also the peak tailing was substantially diminished, giving an ability to separate DNA adducts equal to or better than the TLC method. The method has been applied to 2-nitrofluorene (NF), a carcinogenic air pollutant, and N-acetyl-2-aminofluorene (AAF), a model carcinogen which is also a metabolite of NF. A number of DNA adducts are formed in the livers of rats. After oral administration of AAF and NF, DNA adducts in the liver have been characterized as dG-C8-AF and dG-C8-AAF. The major DNA adduct found in both NF- and AAF-administered animals was dG-C8-AF. The described HPLC method can, with minor adjustments, generally be used to analyze 32P-postlabelled DNA adducts.
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